B细胞转录激活因子对急性哮喘小鼠气道炎症的调控机制

发布时间：2018-07-08 13:01

  本文选题：支气管哮喘 + 白细胞介素17　； 参考：《泸州医学院》2013年硕士论文

【摘要】：目的：探讨B细胞转录激活因子(B cell activating transcription factor, BATF)对卵清蛋白(OVA)致急性哮喘小鼠气道炎症的调控机制及其与维甲酸孤儿核受体γt (RORyt)的关系。方法：(1)将24只健康雌性BALB/c小鼠(SPF级,体重15-18g,周龄68周),随机分为3组,0.9%生理盐水对照组(NS组)、哮喘模型组(AS组)、地塞米松治疗组(DEX组),每组8只。AS组、DEX组小鼠分别于第0、7、14天用卵清蛋白抗原液(100 μg OVA和lmg氢氧化铝混合的生理盐水混悬液)200 ul腹腔内注射致敏,NS组用等体积生理盐水以同种方式代替抗原液。第21天起,NS组以生理盐水,AS组、DEX组以2%的OVA超声雾化吸入进行激发,每天1次,每次40分钟,连续5天。DEX组于每次激发前30分钟,予以地塞米松1. Omg/kg腹腔注射进行干预,NS组、AS组则予以等体积的生理盐水腹腔注射,时间同上。各组小鼠末次激发结束24h后腹腔注射麻醉,行支气管肺泡灌洗及留取肺组织标本。(2)分别对支气管肺泡灌洗液(Bronchoalveolar lavage fluid, BALF)中白细胞(WBC)及嗜酸性粒细胞(EOS)、中性粒细胞(PMN)进行计数；双抗体夹心酶联免疫吸附法(ELISA)检测上清液中白细胞介素17 (Interleukin 17, IL-17)的表达；肺组织病理切片,HE染色观察支气管肺组织炎症浸润程度；实时荧光定量聚合酶链式反应(real time Polymerase Chain Reaction, RT-PCR)分别检测肺组织中IL-17、BATF、 RORγt在mRNA水平的表达情况。利用SPSS17.0软件对各组实验数据进行统计学分析,变量以均数±标准差(x±s)表示,组间变量用单因素方差分析,两变量的相关关系用直线相关分析,统计学差异均以p0.05界定。结果： (1)病理组织切片HE染色结果显示：NS组小鼠支气管管壁平滑肌较薄,管腔光滑、规则,腔内未见分泌物积存,气道黏膜皱襞低平规则,肺泡间隔宽且清晰,支气管血管管周及肺泡间隔内无明显炎症细胞浸润；AS组小鼠支气管管壁平滑肌明显增厚,管腔内大量粘液栓形成及气道黏膜皱襞肥厚增生不规则,纤毛排列紊乱,致支气管管腔极度狭窄,多处气道上皮断裂、脱落不完整,支气管血管管周及肺泡间隔内大量炎症细胞浸润,肺泡间隔增厚明显,分界不清,炎症细胞浸润多以EOS、淋巴细胞及P恻为主；DEX组小鼠支气管管壁增厚及管腔狭窄的程度均较AS组减轻,管腔内分泌物显著减少,肺泡间隔清晰可见,支气管血管管周及肺泡间隔内炎症细胞浸润情况有所改善。(2)AS组BALF中的白细胞总数、嗜酸性粒细胞和中性粒细胞数较NS组明显增多(PO.01)；与AS组相比较,DEX组白细胞总数、嗜酸性粒细胞及中性粒细胞数明显减少(P0.01),但仍比NS组多(P0.05)。 (3)ELISA结果显示：AS组BALF中IL一17蛋白浓度明显高于NS组、DEX组(P0.05)；与AS组相比,DEX组BALF中IL-17蛋白浓度有所降低,差异具有统计学意义(P0.05)。 (4)RT-PCR结果显示：AS组肺组织IL-17、BATF、RORγt mRNA的表达水平明显高于NS组、DEX组,差异均具有统计学意义(PO.05)；与AS组相比,DEX组肺组织IL-17、BATF、RORγt mRNA的表达水平降低,差异具有统计学意义(P0.05)。(5)相关关系分析：小鼠肺组织IL-17 mRNA的表达与BATF、 RORγt的表达及BALF中WBC、EOS、PMN计数呈正相关(r=0.934,2=0.776, r=0.833,2=0.852,2=0.916,p均0.05)；小鼠肺组织BATF与ROR γ t mRNA表达及BALF中WBC、EOS、PMN计数呈明显正相关(r=0.860,r=0.884, r=0.890,r=0.951, p均0.05)。结论： (1)在急性哮喘小鼠支气管肺组织中存在BATF、IL-17、RORγt表达的上调,且三者的表达呈正相关性。(2) BATF的表达增高可能是引起哮喘小鼠气道炎症的关键因素,为临床通过转录因子途径治疗哮喘提供新的理论依据。(3)地塞米松可能是通过下调BATF、RORγt的表达而降低Th17细胞的分化发育及分泌功能,进而减轻气道的炎症性损害及哮喘症状,最终达到抑制气道炎症的效果。
[Abstract]:Objective: To investigate the regulation mechanism of B cell activating transcription factor (BATF) on airway inflammation in acute asthmatic mice induced by ovalbumin (OVA) and its relationship with the orphan retinoic receptor gamma t (RORyt). Methods: (1) 24 healthy female BALB/c mice (SPF grade, body weight, 68 weeks) were randomly divided into 3. Group 0.9%, 0.9% saline control group (group NS), asthma model group (group AS), dexamethasone group (group DEX), 8.AS groups in each group. Group DEX mice were sensitized by intraperitoneal injection of ovalbumin antigen (100 mu g OVA and LMG hydroxide mixed suspension of sodium hydroxide) 200 UL, and NS group with isovolumetric saline in the same way Instead of antigenic fluid. Twenty-first days from twenty-first days, group NS was stimulated with physiological saline, AS group, and group DEX with 2% OVA ultrasonic atomization inhalation, 1 times a day, 40 minutes each time, 30 minutes before each stimulation for 5 days in.DEX group, and dexamethasone 1. Omg/kg intraperitoneal injection, NS group, AS group were given equal volume of saline intraperitoneal injection, time the same. The group of mice at the end of 24h was injected with intraperitoneal injection, and bronchoalveolar lavage and lung tissue were collected. (2) the count of leukocyte (WBC) and eosinophil (EOS), neutrophils (PMN) in bronchoalveolar lavage fluid (Bronchoalveolar lavage fluid, BALF), and double antibody sandwich enzyme-linked immunosorbent assay (ELISA) examination, respectively. The expression of interleukin 17 (Interleukin 17, IL-17) in the supernatant, pathological sections of lung tissue and HE staining were used to observe the degree of inflammatory infiltration in bronchopulmonary tissues, and the real-time fluorescence quantitative polymerase chain reaction (real time Polymerase Chain Reaction, RT-PCR) was used to detect the expression of IL-17, BATF, ROR gamma at the level of lung tissue. SPSS17.0 software was used to make statistical analysis on the experimental data of each group. The variables were expressed by mean mean deviation (x + s). The inter group variables were analyzed by single factor variance. The correlation of the two variables was analyzed with linear correlation. The statistical differences were all defined by P0.05. Results: (1) the HE staining results of pathological sections showed that the bronchial tube wall of group NS mice The smooth muscle was thin, smooth and smooth and regular. There was no secretions in the cavity. The airway mucosa folds were low and regular, the alveolar septum was wide and clear. There was no obvious inflammatory cell infiltration in the peribronchovascular and alveolar septum. The smooth muscle of the bronchial tube wall in the AS group was obviously thickened, a large number of mucous suppositories in the endotracheal cavity and the hypertrophy of the airway mucosa fold increased. Irregular birth, disorder of cilia, severe stenosis of bronchial tube, fracture of airway epithelium, incomplete exfoliation, infiltration of inflammatory cells in peribronchovascular tube and alveolar septum, thickening of alveolar septum, unclear boundary, EOS and P, and thickening of bronchial tube wall in group DEX and the thickening of bronchial tube wall in group of mice. The degree of narrowing of the lumen was less than that in the AS group, the secretion in the lumen decreased significantly, the alveolar septum was clearly visible, and the infiltration of inflammatory cells in the bronchovascular tube and the alveolar septum was improved. (2) the number of white blood cells in the AS group and the number of eosinophils and neutrophils in the BALF group were significantly increased (PO.01); compared with the AS group, the DEX group was compared with the group AS. The number of white blood cells, eosinophils and neutrophils decreased significantly (P0.01), but still more than group NS (P0.05). (3) ELISA results showed that the concentration of IL 1 17 protein in AS group BALF was significantly higher than that of NS group and DEX group (P0.05). The expression level of IL-17, BATF, ROR gamma t mRNA in the lung tissue of AS group was significantly higher than that of the NS group, and the difference in DEX group was statistically significant (PO.05). Compared with the AS group, the expression level of lung tissue in DEX group was reduced. (5) correlation Analysis: expression of lung tissue in mice The expression of ROR gamma t is positively correlated with WBC, EOS, PMN count in BALF (r=0.934,2=0.776, r=0.833,2=0.852,2=0.916, P are 0.05), and there is a significant positive correlation between BATF and ROR t in mice lung tissue. Conclusion: (1) in bronchial and lung tissues of acute asthmatic mice. The expression of BATF, IL-17, ROR gamma t was up-regulated and the expression of the three was positively correlated. (2) the increase of BATF expression may be the key factor in the airway inflammation in asthmatic mice, which provides a new theoretical basis for the treatment of asthma through the transcription factor pathway. (3) dexamethasone may reduce the fraction of Th17 cells by reducing the expression of BATF and ROR gamma t. The development and secretion function can reduce airway inflammation and asthma symptoms, and finally achieve the effect of inhibiting airway inflammation.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R562.25

【参考文献】

中国期刊全文数据库 前1条

1 魏东;刘学东;;支气管肺泡灌洗术治疗难治性重度哮喘的临床分析[J];中国医药科学;2011年15期

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